Methods and compositions for treatment of ocular disorders and blinding diseases

ABSTRACT

Codon optimized nucleic acid sequences for the long form and short form of RdCVF are provided, as well as recombinant viral vectors, such as AAV, expression cassettes, proviral plasmids or other plasmids containing the codon optimized sequences. Recombinant vectors are provided that express the codon optimized RdCVFL and RdCVF individually, or express two copies of a codon optimized RdCVF or RdCVFL nucleic acid sequence, or both RdCVFL and RdCVF in a single vector or virus. Compositions containing these codon optimized sequences are useful in methods for treating, retarding or halting certain blinding diseases resulting from the absence or inappropriate expression of RdCVF and RdCVFL.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“15-7572PCT_SEQ_List_ST25.txt ” and dated Jan. 4, 2017.

BACKGROUND OF THE INVENTION

Rod-cone dystrophies, such as retinitis pigmentosa (RP), are geneticallyheterogeneous retinal degenerative diseases characterized by theprogressive death of rod photoreceptors, followed by the consecutiveloss of cones. RP patients initially present with loss of vision underdim-light conditions as a result of rod dysfunction, with relativepreservation of macular cone-mediated vision. As the disease progresses,however, the primary loss of rods is followed by cone degeneration and adeficit in corresponding cone-mediated vision. Retention ofcone-mediated sight in RP patients would lead to a significantimprovement in their quality of life.

A variety of methods for treatment of such diseases have involved aprotein, termed RdCVF, which is differentially transcribed and expressedin subjects suffering from retinal dystrophies, including age-relatedmacular degeneration. The long (RdCVFL) and short (RdCVF) forms producedby alternative splicing of the NXNL1 gene have been identified in humansand other mammals. See, e.g., US Patent Application Publication No.US2009/0062188; Byrne et al “Viral-mediated RdCVF and RdCVFL expressionprotects cone and rod photoreceptors in retinal degeneration”, January2015, J. Clin. Invest., 125(1):105-116.

No currently approved treatment for retinal degenerations exists otherthan one treatment which involves oral administration of high dosevitamin A. That treatment, however, is controversial and now that weknow more about the retinoid cycle in retinal degenerations, is, infact, likely to worsen retinal degeneration in many genetic forms ofretinal disease.

A continuing need in the art therefore exists for new and effectivetools to facilitate treatment of ocular diseases such as retinaldegenerations, RP, macular degeneration, and other rod-cone dystrophiesand retinal degenerative diseases.

SUMMARY OF THE INVENTION

In one aspect, a codon optimized cDNA sequence SEQ ID NO: 1 encodinghuman RdCVFL long form or a codon optimized cDNA sequence SEQ ID NO: 2encoding RdCVF short form is provided.

In another aspect an expression cassette comprises a codon optimizednucleic acid sequence SEQ ID NO: 1 that encodes RdCVFL or a codonoptimized nucleic acid sequence SEQ ID NO: 2 that encodes RdCVF, or botha codon optimized nucleic acid sequence SEQ ID NO: 2 that encodes RdCVFand a codon optimized nucleic acid sequence SEQ ID NO: 1 that encodesRdCVFL, or two copies of a codon optimized nucleic acid sequence SEQ IDNO: 2 that encodes RdCVF, or two copies of a codon optimized nucleicacid sequence SEQ ID NO: 1 that encodes RdCVFL. In still otherembodiments, the expression cassette is positioned between 5′ and 3′ AAVITR sequences, then referred to as an rAAV genome.

In another aspect, a vector is provided that contains one or more of theexpression cassettes described herein and host cells containing thevectors or expression cassettes are provided.

In another aspect, a proviral plasmid comprises sequences encoding anAAV capsid and an recombinant AAV genome that comprises AAV invertedterminal repeat sequences and an expression cassette comprising thecodon optimized nucleic acid sequence(s) that encodes RdCVFL, RdCVF-S,both RdCVFL and RdCVF, two copies of RdCVF, or two copies of RdCVFL, andexpression control sequences that direct expression of the encodedprotein(s) in a host cell. In certain embodiments, the AAV genome ismodular.

In another embodiment, a recombinant adeno-associated virus (AAV)comprises an AAV capsid and an recombinant AAV genome that comprises AAVinverted terminal repeat sequences and an expression cassette comprisinga codon optimized nucleic acid sequence that encodes RdCVFL, RdCVF, bothRdCVF and RdCVFL, or two copies of RdCVF or two copies of RdCVFL, andexpression control sequences that direct expression of the encodedprotein(s) in a host cell.

In yet a further aspect a pharmaceutical composition comprises apharmaceutically acceptable carrier, diluent, excipient and/or adjuvantand the nucleic acid sequence, a plasmid, a vector, or a viral vector,such as the rAAV, described specifically herein.

In another aspect, a method for treating, retarding or haltingprogression of blindness in a mammalian subject comprises administeringthe compositions described herein containing a codon optimized cDNAsequence encoding human RdCVFL long form or RdCVF short form, or bothforms of RdCVF or multiple copies of the short or long form.

In yet a further aspect, a method of generating a recombinant rAAVcomprises culturing a packaging cell carrying a plasmid or proviralplasmid containing the codon optimized cDNA sequence encoding humanRdCVFL long form or RdCVF short form, or both forms of RdCVF or multiplecopies of the short or long form, in the presence of sufficient viralsequences to permit packaging of the AAV viral genome into an infectiousAAV envelope or capsid.

Still other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO: 1) is a human codon optimized DNA sequence encodingRdCVFL with N-terminal SfiI and Kozak and C-terminal Bg1II restrictionssites added for cloning. Nucleotides 23-655 of SEQ ID NO: 1 representthe sequence of codon optimized RdCVFL. The restriction sites arerepresented by italicized and lower case lettering at nucleotides 1-22and 656-665 of SEQ ID NO: 1.

FIG. 2 (SEQ ID NO: 2) is a human codon optimized DNA sequence encodingRdCVF with N-terminal Notl and Kozak and C-terminal MI restrictionssites added for cloning. Nucleotides 16-339 of SEQ ID NO: 2 representthe sequence of codon optimized RdCVF (short form). The restrictionsites are represented by italicized and lower case lettering atnucleotides 1-15 and 340-348 of SEQ ID NO: 2.

FIG. 3 is the alignment of the long form of optimized RdCVFL(Nucleotides 23-655 of SEQ ID NO: 1 with an added 5′ ATG codon) with thelong form of native RdCVF SEQ ID NO: 3. Identities are 531/636 (83%).SEQ ID NO: 3 is the 636 nucleotide sequence of the native nucleic acidsequence encoding the long form of Homo sapiens nucleoredoxin-like 1(NXNL1), including a start codon ATG at positions 1-3. Nucleotides 1-327of SEQ ID NO: 3 are the short form of native RdCVF. The long form genesequence is also reported at GenBank accession No. NM_138454.1.

FIG. 4 illustrates the alignment of the short form of optimized RdCVF(nucleotides 16-339 of SEQ ID NO: 2 with an added 5′ ATG codon) with theshort form of native RdCVF (nucleotides 1 to 327 of SEQ ID NO: 3).Identities are 271/327 (83%).

FIG. 5 is a schematic map of a single rAAV genome which contains betweena 5′ ITR and 3′ITR, an expression cassette containing tandem transgenes,i.e., the first transgene containing sequences (including a promoter andpoly A sequence) necessary for expression of a codon optimized shortform of RdCVF (optRdCVF) and the second transgene containing sequences(including a promoter and poly A sequence) necessary for expression of acodon optimized long form of RdCVFL (optRdCVFL).

FIG. 6A is a schematic map of a single rAAV genome which containsbetween a 5′ ITR and 3′ITR, an expression cassette containing tandemtransgenes, i.e., the first transgene containing sequences (including apromoter and poly A sequence) necessary for expression of a codonoptimized short form of RdCVF (optRdCVF) and the second transgenecontaining sequences (including a promoter and poly A sequence)necessary for expression of a second copy of the codon optimized shortform of RdCVF (optRdCVF). This rAAV genome is referred to as 2×RdCVF.

FIG. 6B is a more detailed map of the same rAAV genome of FIG. 6A.

DETAILED DESCRIPTION

The methods and compositions described herein involve compositions andmethods for delivering optimized human rod-cone variability factors(hRdCVF) to mammalian subjects for the treatment of ocular disorders,primarily blinding diseases such as rod-cone dystrophies. Thecompositions and methods described herein involve expression cassettes,vectors, recombinant viruses and other compositions for delivery ofmultiple, different versions of the hRdCVF. Such compositions involveboth codon optimization and the assembly of multiple, different versionsof the hRdCVF, i.e., both the long and short forms or multiple copies ofthe same versions of RdCVF (multiple short or multiple long forms) inthe same expression cassette, i.e., as tandem transgenes flanked by asingle pair of ITR sequences within the same AAV genome, or vector orvirus. These features not only increase the efficacy of the product butalso, since a lower dose of reagent is used, increase safety. It isanticipated that this optimization of the transgene cassette couldtheoretically maximize the level of production of the experimentalprotein compared to levels that can be generated using the endogenoussequence.

The compositions and methods described herein, in one embodiment, areuseful to prevent degeneration of cone photoreceptors in differentgenetic forms of retinal degeneration or in degenerative changesassociated with other multi-systemic diseases (for example, diabeticretinopathy in diabetes).

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The definitions contained in this specification areprovided for clarity in describing the components and compositionsherein and are not intended to limit the claimed invention.

NXNL1 is a member of the family rod-cone variability factor genes genesinvolved in a number of ocular diseases. This gene encodes for a longform, RdCVFL and a short form, RdCVF. The native nucleic acid sequenceencoding human RdCVF, e.g., Homo sapiens nucleoredoxin-like 1 (NXNL1),is shown in SEQ ID NO: 3. See also GenBank accession No. NM_138454.1.See, also U.S. Pat. No. 7,795,387; U.S. Pat. No. 8,114,849, U.S. Pat.No. 8,394,756, as well as related patent disclosures, incorporated byreference for additional disclosure of this protein family. The shortform of RdCVF is nucleotides 1-327 of SEQ ID NO: 3.

In certain embodiments of this invention, a subject has an “oculardisorder”, for which the components, compositions and methods of thisinvention are designed to treat. As used herein, the term “subject” asused herein means a mammalian animal, including a human, a veterinary orfarm animal, a domestic animal or pet, and animals normally used forclinical research. In one embodiment, the subject of these methods andcompositions is a human. Still other suitable subjects include, withoutlimitation, murine, rat, canine, feline, porcine, bovine, ovine, andothers. As used herein, the term “subject” is used interchangeably with“patient”.

As used herein “ocular disorder” includes, rod-cone dystrophies andretinal diseases including, without limitation, Stargardt disease(autosomal dominant or autosomal recessive), retinitis pigmentosa,age-related macular degeneration, rod-cone dystrophy, Leber's congenitalamaurosis, Usher's syndrome, Bardet-Biedl Syndrome, Best disease,Bassen-Kornzweig syndrome, retinoschisis, untreated retinal detachment,pattern dystrophy, achromatopsia, choroideremia, ocular albinism,enhanced S cone syndrome, diabetic retinopathy, retinopathy ofprematurity, sickle cell retinopathy, refsun syndrome, CongenitalStationary Night Blindness, glaucoma, gyrate atrophy or retinal veinocclusion. In another embodiment, the subject has, or is at risk ofdeveloping glaucoma, Leber's hereditary optic neuropathy, lysosomalstorage disorder, or peroxisomal disorder. Clinical signs of such oculardiseases include, but are not limited to, decreased peripheral vision,decreased central (reading) vision, decreased night vision, loss ofcolor perception, reduction in visual acuity, decreased photoreceptorfunction, pigmentary changes, and ultimately blindness.

As used herein, the term “treatment” or “treating” is definedencompassing administering to a subject one or more compounds orcompositions described herein for the purposes of amelioration of one ormore symptoms of an ocular disease. “Treatment” can thus include one ormore of reducing onset or progression of an ocular disease, preventingdisease, reducing the severity of the disease symptoms, or retardingtheir progression, including the progression of blindness, removing thedisease symptoms, delaying onset of disease or monitoring progression ofdisease or efficacy of therapy in a given subject.

The term “exogenous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein does not naturally occurin the position in which it exists in a chromosome, recombinant plasmid,vector or host cell. An exogenous nucleic acid sequence also refers to asequence derived from and inserted into the same host cell or subject,but which is present in a non-natural state, e.g. a different copynumber, or under the control of different regulatory elements.

The term “heterologous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein was derived from adifferent organism or a different species of the same organism than thehost cell or subject in which it is expressed. The term “heterologous”when used with reference to a protein or a nucleic acid in a plasmid,expression cassette, or vector, indicates that the protein or thenucleic acid is present with another sequence or subsequence which withwhich the protein or nucleic acid in question is not found in the samerelationship to each other in nature.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the bases in the two sequences which are the samewhen aligned for correspondence. The percent identity is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The length of sequence identity comparison maybe over the full-length of the RdCVF and RdCVFL coding sequence, or afragment of at least about 100 to 150 nucleotides, or as desired.However, identity among smaller fragments, e.g. of at least about ninenucleotides, usually at least about 20 to 24 nucleotides, at least about28 to 32 nucleotides, at least about 36 or more nucleotides, may also bedesired. Multiple sequence alignment programs are also available fornucleic acid sequences. Examples of such programs include, “Clustal W”,“CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which areaccessible through Web Servers on the internet. Other sources for suchprograms are known to those of skill in the art. Alternatively, VectorNTI utilities are also used. There are also a number of algorithms knownin the art that can be used to measure nucleotide sequence identity,including those contained in the programs described above. As anotherexample, polynucleotide sequences can be compared using Fasta™, aprogram in GCG Version 6.1. Commonly available sequence analysissoftware, more specifically, BLAST or analysis tools provided by publicdatabases may also be used.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

By “engineered” is meant that the nucleic acid sequences encoding theRdCVF (short form) and RdCVFL (long form) proteins described herein areassembled and placed into any suitable genetic element, e.g., naked DNA,phage, transposon, cosmid, episome, etc., which transfers the RdCVFsequences carried thereon to a host cell, e.g., for generating non-viraldelivery systems (e.g., RNA-based systems, naked DNA, or the like) orfor generating viral vectors in a packaging host cell and/or fordelivery to a host cells in a subject. In one embodiment, the geneticelement is a plasmid. The methods used to make such engineeredconstructs are known to those with skill in nucleic acid manipulationand include genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Green and Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(2012). The teachings of this specification coupled with knowntechniques permits one of skill in the art to reproduce the exemplifiedThe term “transgene” as used herein means an exogenous or engineeredprotein-encoding nucleic acid sequence that is under the control of apromoter or expression control sequence in an expression cassette, rAAVgenome, recombinant plasmid or proviral plasmid, vector, or host celldescribed in this specification. In certain embodiments, the transgeneis a codon optimized RdCVFL encoding sequence SEQ ID NO: 1. In certainembodiments, the transgene is a codon optimized RdCVF (short form)encoding sequence SEQ ID NO:2. In other embodiments, both codonoptimized and natural RdCVF and RdCVFL encoding sequences, in variouscombinations serve as the transgene.

A “vector” as used herein is a nucleic acid molecule into which anexogenous or heterologous or engineered nucleic acid transgene may beinserted which can then be introduced into an appropriate host cell.Vectors preferably have one or more origin of replication, and one ormore site into which the recombinant DNA can be inserted. Vectors oftenhave convenient means by which cells with vectors can be selected fromthose without, e.g., they encode drug resistance genes. Common vectorsinclude plasmids, viral genomes, and (primarily in yeast and bacteria)“artificial chromosomes.” “Virus vectors” are defined as replicationdefective viruses containing the exogenous or heterologous RdCVF andRdCVFL nucleic acid transgene(s). In one embodiment a expressioncassette as described herein may be engineered onto a plasmid which isused for drug delivery or for production of a viral vector. Suitableviral vectors are preferably replication defective and selected fromamongst those which target ocular cells. Viral vectors may include anyvirus suitable for gene therapy may be used, including but not limitedto adenovirus; herpes virus; lentivirus; retrovirus; parvovirus, etc.However, for ease of understanding, the adeno-associated virus isreferenced herein as an exemplary virus vector.

A “replication-defective virus” or “viral vector” refers to a syntheticor recombinant viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless”-containing only the transgene of interest flanked by thesignals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication.

In still another embodiment, the expression cassette, including any ofthose described herein is employed to generate a recombinant AAV genome.

As used herein, the term “host cell” may refer to the packaging cellline in which a recombinant AAV is produced from a proviral plasmid. Inthe alternative, the term “host cell” may refer to any target cell inwhich expression of the transgene is desired. Thus, a “host cell,”refers to a prokaryotic or eukaryotic cell that contains exogenous orheterologous DNA that has been introduced into the cell by any means,e.g., electroporation, calcium phosphate precipitation, microinjection,transformation, viral infection, transfection, liposome delivery,membrane fusion techniques, high velocity DNA-coated pellets, viralinfection and protoplast fusion.

In certain embodiments herein, the term “host cell” refers to culturesof ocular cells of various mammalian species for in vitro assessment ofthe compositions described herein. In other embodiments herein, the term“host cell” refers to the cells employed to generate and package theviral vector or recombinant virus. Still in other embodiments, the term“host cell” is intended to reference the ocular cells of the subjectbeing treated in vivo for the ocular disease.

As used herein, the term “ocular cells” refers to any cell in, orassociated with the function of, the eye. The term may refer to any oneof photoreceptor cells, including rod, cone and photosensitive ganglioncells or retinal pigment epithelium (RPE) cells. In one embodiment, theocular cells are the photoreceptor cells.

“Plasmids” generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

As used herein, the term “transcriptional control sequence” or“expression control sequence” refers to DNA sequences, such as initiatorsequences, enhancer sequences, and promoter sequences, which induce,repress, or otherwise control the transcription of protein encodingnucleic acid sequences to which they are operably linked.

As used herein, the term “operably linked” or “operatively associated”refers to both expression control sequences that are contiguous with thenucleic acid sequence encoding the RdCVF and RdCVFL and/or expressioncontrol sequences that act in trans or at a distance to control thetranscription and expression thereof.

The term “AAV” or “AAV serotype” as used herein refers to the more than30 naturally occurring and available adeno-associated viruses, as wellas artificial AAVs. Among the AAVs isolated or engineered from human ornon-human primates (NHP) and well characterized, human AAV2 is the firstAAV that was developed as a gene transfer vector; it has been widelyused for efficient gene transfer experiments in different target tissuesand animal models. Unless otherwise specified, the AAV capsid, ITRs, andother selected AAV components described herein, may be readily selectedfrom among any AAV, including, without limitation, AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2,rh8, rh.10, variants of any of the known or mentioned AAVs or AAVs yetto be discovered or variants or mixtures thereof. See, e.g., WO2005/033321. The ITRs or other AAV components may be readily isolated orengineered using techniques available to those of skill in the art froman AAV. Such AAV may be isolated, engineered, or obtained from academic,commercial, or public sources (e.g., the American Type CultureCollection, Manassas, Va.). Alternatively, the AAV sequences may beengineered through synthetic or other suitable means by reference topublished sequences such as are available in the literature or indatabases such as, e.g., GenBank, PubMed, or the like. AAV viruses maybe engineered by conventional molecular biology techniques, making itpossible to optimize these particles for cell specific delivery ofnucleic acid sequences, for minimizing immunogenicity, for tuningstability and particle lifetime, for efficient degradation, for accuratedelivery to the nucleus, etc.

As used herein, “artificial AAV” means, without limitation, an AAV witha non-naturally occurring capsid protein. Such an artificial capsid maybe generated by any suitable technique, using a selected AAV sequence(e.g., a fragment of a vp1 capsid protein) in combination withheterologous sequences which may be obtained from a different selectedAAV, non-contiguous portions of the same AAV, from a non-AAV viralsource, or from a non-viral source. An artificial AAV may be, withoutlimitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAVcapsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein thecapsid of one AAV is replaced with a heterologous capsid protein, areuseful in the invention. In one embodiment, AAV2/5 and AAV2/8 areexemplary pseudotyped vectors.

“Self-complementary AAV” refers a plasmid or vector having an expressioncassette in which a coding region carried by a recombinant AAV nucleicacid sequence has been designed to form an intra-moleculardouble-stranded DNA template. Upon infection, rather than waiting forcell mediated synthesis of the second strand, the two complementaryhalves of scAAV will associate to form one double stranded DNA (dsDNA)unit that is ready for immediate replication and transcription. See,e.g., D M McCarty et al, “Self-complementary recombinantadeno-associated virus (scAAV) vectors promote efficient transductionindependently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8,Number 16, Pages 1248-1254. Self-complementary AAVs are described in,e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of whichis incorporated herein by reference in its entirety.

By “administering” as used in the methods means delivering thecomposition to the target selected cell which is characterized by theocular disease. In one embodiment, the method involves delivering thecomposition by subretinal injection to the photoreceptor cells or otherocular cells. In another embodiment, intravitreal injection to ocularcells is employed. In still another method, injection via the palpebralvein to ocular cells may be employed. Still other methods ofadministration may be selected by one of skill in the art given thisdisclosure. By “administering” or “route of administration” is deliveryof composition described herein, with or without a pharmaceuticalcarrier or excipient, of the subject. Routes of administration may becombined, if desired. In some embodiments, the administration isrepeated periodically. The pharmaceutical compositions described hereinare designed for delivery to subjects in need thereof by any suitableroute or a combination of different routes. Direct delivery to the eye(optionally via ocular delivery, intra-retinal injection, intravitreal,topical), or delivery via systemic routes, e.g., intraarterial,intraocular, intravenous, intramuscular, subcutaneous, intradermal, andother parental routes of administration. The nucleic acid moleculesand/or vectors described herein may be delivered in a single compositionor multiple compositions. Optionally, two or more different AAV may bedelivered, or multiple viruses [see, e.g., WO20 2011/126808 and WO2013/049493]. In another embodiment, multiple viruses may containdifferent replication-defective viruses (e.g., AAV and adenovirus),alone or in combination with proteins.

The terms “a” or “an” refers to one or more, for example, “an inhibitor”is understood to represent one or more such compounds, molecules,peptides or antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” are used interchangeably herein.

As used herein, the term “about” means a variability of plus or minus10% from the reference given, unless otherwise specified.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively, i.e., to include otherunspecified components or process steps. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively, i.e., to exclude components or steps notspecifically recited.

Certain compositions described herein are isolated, or synthetically orrecombinantly engineered nucleic acid sequences that provide novelcodon-optimized sequences encoding hRdCVFL (long form) and hRdCVF (shortform). In one embodiment, an isolated or engineered codon optimizednucleic acid sequence encoding human RdCVFL long form is provided. Thiscodon-optimized RdCVFL SEQ ID NO: 1 contains an N-terminal SfiI andKozak and C-terminal Bg1II restrictions sites added for cloning. Whenaligned with the native nucleic acid sequence, the codon optimizedRdCVFL may have a percent identity of at least 50%, or at least 60%, orat least 70%, or at least 80% or at least 90%, including any integerbetween any of those ranges. In one embodiment, the codon optimizedRdCVFL has a percent identify with the native sequence of at least 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. In one embodiment,when aligned with the native nucleic acid sequence SEQ ID NO: 3, it isrevealed that codon optimized RdCVFL SEQ ID NO: 1 has a percent sequenceidentity of only 83% (see FIG. 3).

In another embodiment, an isolated codon optimized nucleic acid sequenceencoding human RdCVF short form is provided. When aligned with thenative nucleic acid sequence, the codon optimized RdCVF may have apercent identity of at least 50%, or at least 60%, or at least 70%, orat least 80% or at least 90%, including any integer between any of thoseranges. In one embodiment, the codon optimized RdCVF has a percentidentify with the native sequence of at least 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99%. In one embodiment, the codon-optimizedRdCVF SEQ ID NO: 2 contains N-terminal Notl and Kozak and C-terminalBc1I restrictions sites added for cloning. When aligned with the nativenucleic acid sequence (nucleotides 1-327 of SEQ ID NO: 3), it isrevealed that the encoding sequence of SEQ ID NO: 2 has a percentsequence identity of only 83% with the short form of the native sequence(see FIG. 4).

In one embodiment, the optimized nucleic acid sequences encoding thehRdCVF long and/or short constructs described herein are engineered intoany suitable genetic element, e.g., naked DNA, phage, transposon,cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers theRdCVF sequences carried thereon to a host cell, e.g., for generatingnanoparticles carrying DNA or RNA, viral vectors in a packaging hostcell and/or for delivery to a host cells in subject. In one embodiment,the genetic element is a plasmid.

The selected genetic element may be delivered by any suitable method,including transfection, electroporation, liposome delivery, membranefusion techniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. The methods used to make such constructs are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (2012).

A variety of expression cassettes are provided which employ SEQ ID NOs.1 and 2 for expression of multiple or different versions of the hRdCVFprotein. As used herein, an “expression cassette” refers to a nucleicacid molecule which comprises coding sequences for the optimized RdCVFLand/or RdCVF (short) proteins, promoter, and may include otherregulatory sequences therefor, which cassette may be engineered into agenetic element or plasmid, and/or packaged into the capsid of a viralvector (e.g., a viral particle). In one embodiment, an expressioncassette comprises a codon optimized nucleic acid sequence, i.e., SEQ IDNO: 1, that encodes RdCVFL. In one embodiment, the cassette provides thecodon optimized RdCVFL operatively associated with expression controlsequences that direct expression of the codon optimized nucleic acidsequence that encodes RdCVFL in a host cell.

In another embodiment, an expression cassette comprises a codonoptimized nucleic acid sequence, i.e., SEQ ID NO: 2, that encodes RdCVF.In one embodiment, the cassette provides the codon optimized RdCVFoperatively associated with expression control sequences that directexpression of the codon optimized nucleic acid sequence that encodesRdCVF in a host cell.

In still another embodiment, an expression cassette comprises a codonoptimized nucleic acid sequence that encodes RdCVFL and RdCVF. In oneembodiment of such an expression cassette, the sequence encoding RdCVFLis operatively associated with the a first expression controlsequence(s) that direct expression of the codon optimized nucleic acidsequence that encodes RdCVFL in a host cell (a first transgene) and thesequence encoding RdCVF is operatively associated with the a secondexpression control sequence(s) that direct expression of the codonoptimized nucleic acid sequence that encodes RdCVF in a host cell (asecond transgene). Transcription of each optimized sequence iscontrolled by an independent expression control sequence and thetransgenes are in tandem orientation within a single rAAV genome orexpression cassette

In one embodiment, the second expression control sequence(s) are thecopies of, but independent from, the first expression controlsequence(s). In another embodiment, the second expression controlsequence(s) are completely different and independent from, the firstexpression control sequence(s). In yet another embodiment, a singleexpression control sequence is operatively associated with bothoptimized sequences, so that both sequences are expressed at the sametime under the same control sequences. In another embodiment, the twooptimized sequences are expressed as a fusion sequence.

Further in one embodiment, the expression cassette comprises the hRdCVFLsequence under control of the first expression control sequence inposition 5′ to the hRdCVF sequence, which is under control of the secondexpression control sequence. In another embodiment, the expressioncassette comprises the hRdCVF sequence under control of the secondexpression control sequence in position 5′ to the hRdCVFL sequence,which is under control of the first expression control sequence. In yetanother embodiment, where the expression cassette contains a singleexpression control sequence for control of transcription of bothoptimized sequences, the hRdCVF sequence is in position 5′ to thehRdCVFL sequence or the hRdCVFL sequence is in position 5′ to the hRdCVFsequence. In still other embodiments, the hRdCVF and hRdCVFL sequencesmay be in position to be expressed as a fusion protein.

In still another embodiment, an expression cassette comprises multiplecopies of the RdCVF sequences, in which at one one copy is the codonoptimized nucleic acid sequence, i.e., SEQ ID NO: 2, that encodes RdCVF.In one embodiment of such an expression cassette, the sequence encodingcodon optimized RdCVF is operatively associated with the a firstexpression control sequence(s) that direct expression of the codonoptimized nucleic acid sequence that encodes one copy of RdCVF in a hostcell and the sequence encoding the second copy of codon-optimized RdCVFis operatively associated with a second expression control sequence(s)that direct expression of the codon optimized nucleic acid sequence thatencodes the second copy of RdCVF in a host cell, i.e., transcription ofeach optimized sequence is controlled by an independent expressioncontrol sequence. In one embodiment, the second expression controlsequence(s) are the copies of, but independent from, the firstexpression control sequence(s). In another embodiment, the secondexpression control sequence(s) are completely different and independentfrom, the first expression control sequence(s).

Similarly, in still another embodiment, an expression cassette comprisesmultiple copies of the RdCVFL sequences, in which at one one copy is thecodon optimized nucleic acid sequence, i.e., SEQ ID NO: 1, that encodesRdCVFL. In one embodiment of such an expression cassette, the sequenceencoding codon optimized RdCVFL is operatively associated with the afirst expression control sequence(s) that direct expression of the codonoptimized nucleic acid sequence that encodes one copy of RdCVFL in ahost cell and the sequence encoding the second copy of codon-optimizedRdCVFL is operatively associated with a second expression controlsequence(s) that direct expression of the codon optimized nucleic acidsequence that encodes the second copy of RdCVFL in a host cell, i.e.,transcription of each optimized sequence is controlled by an independentexpression control sequence. In one embodiment, the second expressioncontrol sequence(s) are the copies of, but independent from, the firstexpression control sequence(s). In another embodiment, the secondexpression control sequence(s) are completely different and independentfrom, the first expression control sequence(s).

In yet another embodiment, a single expression control sequence isoperatively associated with both optimized sequences, so that bothsequences are expressed at the same time under the same controlsequences. In another embodiment, the two optimized sequences areexpressed as a fusion sequence. In still other embodiments, theoptimized RdCVF is present in the expression cassette with a nativeversion of the RdCVF sequence.

As described above for the expression cassettes containing both RdCVFand RdCVFL, in embodiments in which two different versions of RdCVF areemployed, the codon optimized sequence may be positioned, 5′ or 3′ toanother version of the short sequences. One of skill in the art mayreadily design constructs similar to those of the Examples below in viewof the teachings of this specification.

As described herein, a “rAAV genome” is meant to describe an expressioncassette or an expression cassette containing tandem transgenes, asdescribed herein flanked on its 5′ end by a 5′AAV inverted terminalrepeat sequence (ITR) and on its 3′ end by a 3′ AAV ITR. Thus, this rAAVgenome contains the minimal sequences required to package the expressioncassette into an AAV viral particle, i.e., the AAV 5′ and 3′ ITRs. TheAAV ITRs may be obtained from the ITR sequences of any AAV, such asdescribed herein. These ITRs may be of the same AAV origin as the capsidemployed in the resulting recombinant AAV, or of a different AAV origin(to produce an AAV pseudotype). In one embodiment, the ITR sequencesfrom AAV2, or the deleted version thereof (AITR), are used forconvenience and to accelerate regulatory approval. However, ITRs fromother AAV sources may be selected. Each rAAV genome can be thenintroduced into a proviral plasmid following the teachings ofWO2012/158757. The proviral plasmids are cultured in the host cellswhich express the AAV cap and/or rep proteins. In the host cells, eachrAAV genome is rescued and packaged into the capsid protein or envelopeprotein to form an infectious viral particle.

In yet another embodiment, a vector comprising any of the expressioncassettes described herein is provided. As described above, such vectorscan be plasmids of variety of origins and are useful in certainembodiments for the generation of recombinant replication defectiveviruses as described further herein.

In one another embodiment, the vector is a proviral plasmid thatcomprises an AAV capsid and an recombinant AAV genome, wherein said rAAVgenome comprises AAV inverted terminal repeat sequences and anexpression cassette as described above comprising a codon optimizednucleic acid sequence that encodes RdCVFL, RdCVF, both RdCVFL and RdCVFor multiple (i.e., at least two) copies of RdCVF or two copies ofRdCVFL, and expression control sequences that direct expression of theencoded protein in a host cell.

One type of proviral plasmid comprises a modular recombinant AAV genomethat permits portions of the components of the rAAV genome to be removedand repeatedly replaced with other components without destroying therestriction sites in the plasmid. Such a proviral plasmid is one thatcontains a 5′ AAV ITR sequence, the ITR flanked upstream by restrictionsite 1 and downstream by restriction site 2; a selected promoter flankedupstream by restriction site 2 and downstream by restriction site 3.Another component of the modular rAAV is a polylinker sequencecomprising at least restriction site 3, restriction site 4 andrestriction site 5, that contains a codon optimized nucleic acidsequence that encodes RdCVFL, a codon optimized nucleic acid sequencethat encodes RdCVF, a codon optimized nucleic acid sequence that encodesRdCVFL and a codon optimized nucleic acid sequence that encodes RdCVF,or two or more copies of a sequence that encodes RdCVF, at least onesuch sequence being a codon optimized nucleic acid sequence encodingRdCVF. The RdCVF encoding sequences are located between any two of therestriction sites 3, 4 and 5, and are operatively linked to, and underthe regulatory control of, the promoter. Alternatively, the secondencoding sequence is inserted into the polylinker sequence along withthe second expression control sequence of the expression cassette asdescribed above.

Additional components of the modular rAAV include a polyadenylationsequence flanked upstream by restriction site 4 or 5 and downstream byrestriction site 6; and a 3′ AAV ITR sequence flanked upstream byrestriction site 6 and downstream by restriction site 7. The proviralplasmid also contains elements necessary for replication in bacterialcells, and a resistance gene. Each of the above-noted restriction sites1 through 7 occurs only once in the proviral plasmid and is cleaved by adifferent enzyme that cannot cleave another restriction site in theplasmid and thereby permit independent and repeated removal, replacementor substitution of the entire rAAV modular genome or only the elementsflanked by those restriction sites from the plasmid. Such plasmids aredescribed in detail in International Patent Application Publication No.WO2012/158757, incorporated by reference herein.

In still a further embodiment, a recombinant adeno-associated virus(AAV) vector is provided for delivery of the RdCVF constructs andoptimized sequences described herein. An adeno-associated virus (AAV)viral vector is an AAV DNase-resistant particle having an AAV proteincapsid into which is packaged nucleic acid sequences for delivery totarget cells. An AAV capsid is composed of 60 capsid (cap) proteinsubunits, VP1, VP2, and VP3, that are arranged in an icosahedralsymmetry in a ratio of approximately 1:1:10 to 1:1:20, depending uponthe selected AAV. AAVs may be selected as sources for capsids of AAVviral vectors as identified above. See, e.g., US Published PatentApplication No. 2007-0036760-A1; US Published Patent Application No.2009-0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV7 and othersimian AAV), U.S. Pat. No. 7,790,449 and U.S. Pat. No. 7,282,199 (AAV8),WO 2005/033321 and U.S. Pat. No. 7,906,111 (AAV9), and WO 2006/110689,and WO 2003/042397 (rh.10). These documents also describe other AAVwhich may be selected for generating AAV and are incorporated byreference. In some embodiments, an AAV cap for use in the viral vectorcan be generated by mutagenesis (i.e., by insertions, deletions, orsubstitutions) of one of the aforementioned AAV capsids or its encodingnucleic acid. In some embodiments, the AAV capsid is chimeric,comprising domains from two or three or four or more of theaforementioned AAV capsid proteins. In some embodiments, the AAV capsidis a mosaic of Vp1, Vp2, and Vp3 monomers from two or three differentAAVs or recombinant AAVs. In some embodiments, an rAAV compositioncomprises more than one of the aforementioned Caps.

In another embodiment, the AAV capsid includes variants which mayinclude up to about 10% variation from any described or known AAV capsidsequence. That is, the AAV capsid shares about 90% identity to about99.9% identity, about 95% to about 99% identity or about 97% to about98% identity to an AAV capsid provided herein and/or known in the art.In one embodiment, the AAV capsid shares at least 95% identity with anAAV capsid. When determining the percent identity of an AAV capsid, thecomparison may be made over any of the variable proteins (e.g., vp1,vp2, or vp3). In one embodiment, the AAV capsid shares at least 95%identity with the AAV8 vp3. In another embodiment, a self-complementaryAAV is used.

For packaging an expression cassette or rAAV genome or proviral plasmidinto virions, the ITRs are the only AAV components required in cis inthe same construct as the transgene. In one embodiment, the codingsequences for the replication (rep) and/or capsid (cap) are removed fromthe AAV genome and supplied in trans or by a packaging cell line inorder to generate the AAV vector. For example, as described above, apseudotyped AAV may contain ITRs from a source which differs from thesource of the AAV capsid. Additionally or alternatively, a chimeric AAVcapsid may be utilized. Still other AAV components may be selected.Sources of such AAV sequences are described herein and may also beisolated or engineered obtained from academic, commercial, or publicsources (e.g., the American Type Culture Collection, Manassas, Va.).Alternatively, the AAV sequences may be obtained through synthetic orother suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank®,PubMed®, or the like.

Methods for generating and isolating AAV viral vectors suitable fordelivery to a subject are known in the art. See, e.g., U.S. Pat. No.7,790,449; U.S. Pat. No. 7,282,199; WO 2003/042397; WO 2005/033321, WO2006/110689; and U.S. Pat. No. 7,588,772 B2. In a one system, a producercell line is transiently transfected with a construct that encodes thetransgene flanked by ITRs and a construct(s) that encodes rep and cap.In a second system, a packaging cell line that stably supplies rep andcap is transiently transfected with a construct encoding the transgeneflanked by ITRs. In each of these systems, AAV virions are produced inresponse to infection with helper adenovirus or herpesvirus, requiringthe separation of the rAAVs from contaminating virus. More recently,systems have been developed that do not require infection with helpervirus to recover the AAV—the required helper functions (i.e., adenovirusE1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, andherpesvirus polymerase) are also supplied, in trans, by the system. Inthese newer systems, the helper functions can be supplied by transienttransfection of the cells with constructs that encode the requiredhelper functions, or the cells can be engineered to stably contain genesencoding the helper functions, the expression of which can be controlledat the transcriptional or posttranscriptional level.

In yet another system, the transgene flanked by ITRs and rep/cap genesare introduced into insect cells by infection with baculovirus-basedvectors. For reviews on these production systems, see generally, e.g.,Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid forlarge-scale recombinant adeno-associated virus production,” Human GeneTherapy 20:922-929, the contents of each of which is incorporated hereinby reference in its entirety. Methods of making and using these andother AAV production systems are also described in the following U.S.patents, the contents of each of which is incorporated herein byreference in its entirety: U.S. Pat. Nos. 5,139,941; 5,741,683;6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753;7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. Seegenerally, e.g., Grieger & Samulski, 2005, “Adeno-associated virus as agene therapy vector: Vector development, production and clinicalapplications,” Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning etal., 2008, “Recent developments in adeno- associated virus vectortechnology,” J. Gene Med. 10:717-733; and the references cited below,each of which is incorporated herein by reference in its entirety.

The methods used to construct any embodiment of this invention are knownto those with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012). Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Pat. No.5,478,745.

The rAAV vectors comprise an AAV capsid and an recombinant AAV genome,such as described above. In certain embodiments, the rAAV genomecomprises AAV inverted terminal repeat sequences and an expressioncassette comprising a codon optimized nucleic acid sequence that encodesRdCVFL, RdCVF, both RdCVFL and RdCVF or at least two copies of RdCVF, ortwo copies of RdCVFL, with at least one copy optimized or two copiesoptimized, and expression control sequences that direct expression ofthe encoded proteins in a host cell. The rAAV, in other embodiments,further comprises one or more of an intron, a Kozak sequence, a poly A,and post-transcriptional regulatory elements. Such rAAV vectors for usein pharmaceutical compositions for delivery to the eye, may employ acapsid from any of the many known AAVs identified above.

Other conventional components of the expression cassettes, rAAV genomes,and vectors include other components that can be optimized for aspecific species using techniques known in the art including, e.g.,codon optimization, as described herein. The components of thecassettes, vectors, plasmids and viruses or other compositions describedherein include a promoter sequence as part of the expression controlsequences. In one embodiment, a suitable promoter is a hybrid chicken(β-actin (CBA)promoter with cytomegalovirus (CMV) enhancer elements,such as the promoter used in the examples below and represented by thenucleic acid sequences of Tables 1 and 2A and 2B, i.e., nucleotides307-1578 of SEQ ID NO: 5. Still other suitable promoters are the CB7promoter, as well such as viral promoters, constitutive promoters,regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], ora promoter responsive to physiologic cues may be used may be utilized inthe expression cassette, rAAV genomes, vectors, plasmids and virusesdescribed herein.

In another embodiment, the promoter is cell-specific. The term“cell-specific” means that the particular promoter selected for therecombinant vector can direct expression of the optimized RdCVFtransgene in a particular ocular cell type. In one embodiment, thepromoter is specific for expression of the transgene in photoreceptorcells. In another embodiment, the promoter is specific for expression inthe rods and cones. In another embodiment, the promoter is specific forexpression in the rods. In another embodiment, the promoter is specificfor expression in the cones.

Exemplary promoters may be the human G-protein-coupled receptor proteinkinase 1 (GRK1) promoter (Genbank Accession number AY327580). In anotherembodiment, the promoter is a 292 nt fragment (positions 1793-2087) ofthe GRK1 promoter (See, Beltran et al, Gene Therapy 2010 17:1162-74,which is hereby incorporated by reference herein). In another preferredembodiment, the promoter is the human interphotoreceptorretinoid-binding protein proximal (IRBP) promoter. In one embodiment,the promoter is a 235 nt fragment of the hIRBP promoter. In oneembodiment, the promoter is the RPGR proximal promoter (Shu et al, IOVS,May 2102, which is incorporated by reference herein). Other promotersuseful in the invention include, without limitation, the rod opsinpromoter, the red-green opsin promoter, the blue opsin promoter, thecGMP-r3-phosphodiesterase promoter, the mouse opsin promoter (Beltran etal 2010 cited above), the rhodopsin promoter (Mussolino et al, GeneTher, July 2011, 18(7):637-45); the alpha-subunit of cone transducin(Morrissey et al, BMC Dev, Biol, January 2011, 11:3); betaphosphodiesterase (PDE) promoter; the retinitis pigmentosa (RP1)promoter (Nicord et al, J. Gene Med, December 2007, 9(12):1015-23); theNXNL2/NXNL1 promoter (Lambard et al, PLoS One, October 2010,5(10):e13025), the RPE65 promoter; the retinal degenerationslow/peripherin 2 (Rds/perph2) promoter (Cai et al, Exp Eye Res. 2010August; 91(2):186-94); and the VMD2 promoter (Kachi et al, Human GeneTherapy, 2009 (20:31-9)). Each of these documents is incorporated byreference herein. In one embodiment, the promoter is of a small size,under 1000 bp, due to the size limitations of the AAV vector. In anotherembodiment, the promoter is under 400 bp. Other promoters may beselected by one of skill in the art.

In other embodiments, the cassette, vector, plasmid and virus constructsdescribed herein contain other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (poly A) signals; TATA sequences;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); introns;sequences that enhance protein stability; and when desired, sequencesthat enhance secretion of the encoded product. The expression cassetteor vector may contain none, one or more of any of the elements describedherein. Examples of suitable poly A sequences include, e.g., SV40,bovine growth hormone (bGH), and TK poly A. Examples of suitableenhancers include, e.g., the CMV enhancer, the RSV enhancer, the alphafetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-bindingglobulin promoter/alpha1-microglobulin/bikunin enhancer), amongstothers.

In yet other aspects, these nucleic acid sequences, vectors, rAAVgenomes and rAAV viral vectors are useful in a pharmaceuticalcomposition, which also comprises a pharmaceutically acceptable carrier.Such pharmaceutical compositions are used to express the optimizedRdCVFL or RdCVF, or multiple copies of RdCVF or both proteins in theocular cells through delivery by such recombinantly engineered AAVs orartificial AAV's.

To prepare these pharmaceutical compositions containing the nucleic acidsequences, vectors, rAAV genomes and rAAV viral vectors, the sequencesor vectors or viral vector is preferably assessed for contamination byconventional methods and then formulated into a pharmaceuticalcomposition suitable for administration to the eye. Such formulationinvolves the use of a pharmaceutically and/or physiologically acceptablevehicle or carrier, particularly one suitable for administration to theeye, such as buffered saline or other buffers, e.g., HEPES, to maintainpH at appropriate physiological levels, and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. Exemplary physiologically acceptable carriers include sterile,pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.A variety of such known carriers are provided in U.S. Pat. PublicationNo. 7,629,322, incorporated herein by reference. In one embodiment, thecarrier is an isotonic sodium chloride solution. In another embodiment,the carrier is balanced salt solution. In one embodiment, the carrierincludes tween. If the virus is to be stored long-term, it may be frozenin the presence of glycerol or Tween20.

In one exemplary specific embodiment, the composition of the carrier orexcipient contains 180 mM NaCl, 10 mM NaPi, pH 7.3 with 0.0001%-0.01%Pluronic F68 (PF68). The exact composition of the saline component ofthe buffer ranges from 160 mM to 180 mM NaCl. Optional a different pHbuffer (potentially HEPEs, sodium bicarbonate, TRIS) is used in place ofthe buffer specifically described. Still alternatively, a buffercontaining 0.9% NaCl is useful.

Optionally, the compositions of the invention may contain, in additionto the rAAV and/or variants and carrier(s), other conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The pharmaceutical compositions containing the replication-defectiverAAV viruses can be formulated with a physiologically acceptable carrierfor use in gene transfer and gene therapy applications. In the case ofAAV viral vectors, quantification of the genome copies (“GC”), vectorgenomes, or virus particles may be used as the measure of the dosecontained in the formulation or suspension. Any method known in the artcan be used to determine the genome copy (GC) number of thereplication-defective virus compositions of the invention. One methodfor performing AAV GC number titration is as follows: Purified AAVvector samples are first treated with DNase to eliminate un-encapsidatedAAV genome DNA or contaminating plasmid DNA from the production process.The DNase resistant particles are then subjected to heat treatment torelease the genome from the capsid. The released genomes are thenquantitated by real-time PCR using primer/probe sets targeting specificregion of the viral genome (usually poly A signal). In another methodthe effective dose of a recombinant adeno-associated virus carrying anucleic acid sequence encoding the optimized RdCVF transgene under thedesirably are measured as described in S. K. McLaughlin et al, 1988 J.Virol., 62:1963.

As used herein, the term “dosage” can refer to the total dosagedelivered to the subject in the course of treatment, or the amountdelivered in a single unit (or multiple unit or split dosage)administration. The pharmaceutical virus compositions can be formulatedin dosage units to contain an amount of replication-defective viruscarrying the codon optimized nucleic acid sequences encoding hRdCVFand/or hRdCVFL as described herein that is in the range of about 1.0×10⁹GC to about 1.0×10¹⁵ GC including all integers or fractional amountswithin the range. In one embodiment, the compositions are formulated tocontain at least 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,or 9×10⁹ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, or 9×10¹⁰ GC per dose including all integers or fractionalamounts within the range. In another embodiment, the compositions areformulated to contain at least 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, or 9×10¹¹ GC per dose including all integers orfractional amounts within the range. In another embodiment, thecompositions are formulated to contain at least 1×10¹², 2×10¹², 3×10¹²,4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², or 9×10¹² GC per dose includingall integers or fractional amounts within the range. In anotherembodiment, the compositions are formulated to contain at least 1×10¹³,2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, or 9×10¹³ GC perdose including all integers or fractional amounts within the range. Inanother embodiment, the compositions are formulated to contain at least1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, or9×10¹⁴ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵,8×10¹⁵, or 9×10¹⁵ GC per dose including all integers or fractionalamounts within the range. In one embodiment, for human application thedose can range from 1×10¹⁰ to about 1×10¹² GC per dose including allintegers or fractional amounts within the range.

These above doses may be administered in a variety of volumes ofcarrier, excipient or buffer formulation, ranging from about 25 to about1000 microliters, including all numbers within the range, depending onthe size of the area to be treated, the viral titer used, the route ofadministration, and the desired effect of the method. In one embodiment,the volume of carrier, excipient or buffer is at least about 25 μl. Inone embodiment, the volume is about 50 μl. In another embodiment, thevolume is about 75 μl. In another embodiment, the volume is about 100μl. In another embodiment, the volume is about 125 μl. In anotherembodiment, the volume is about 150 μl. In another embodiment, thevolume is about 175 μl. In yet another embodiment, the volume is about2004. In another embodiment, the volume is about 225 μl. In yet anotherembodiment, the volume is about 250 μl. In yet another embodiment, thevolume is about 275 μl. In yet another embodiment, the volume is about300 4. In yet another embodiment, the volume is about 325 μL. In anotherembodiment, the volume is about 350 μl. In another embodiment, thevolume is about 375 μl. In another embodiment, the volume is about 400μl. In another embodiment, the volume is about 450 μl. In anotherembodiment, the volume is about 500 μl. In another embodiment, thevolume is about 550 μl. In another embodiment, the volume is about 600μl. In another embodiment, the volume is about 650 μl. In anotherembodiment, the volume is about 700 μl. In another embodiment, thevolume is between about 700 and 1000 μl.

In one embodiment, the viral constructs may be delivered inconcentrations of from at least least 1×10⁹to about least 1×10¹¹GCs involumes of about 1 μl to about 3 μl for small animal subjects, such asmice. For larger veterinary subjects having eyes about the same size ashuman eyes, the larger human dosages and volumes stated above areuseful. See, e.g., Diehl et al, J. Applied Toxicology, 21:15-23 (2001)for a discussion of good practices for administration of substances tovarious veterinary animals. This document is incorporated herein byreference.

It is desirable that the lowest effective concentration of virus orother delivery vehicle be utilized in order to reduce the risk ofundesirable effects, such as toxicity, retinal dysplasia and detachment.Still other dosages in these ranges may be selected by the attendingphysician, taking into account the physical state of the subject,preferably human, being treated, the age of the subject, the particularocular disorder and the degree to which the disorder, if progressive,has developed.

Yet another aspect described herein is a method for treating, retardingor halting progression of blindness in a mammalian subject having one ormore of the ocular diseases described above, such as rod-conedystrophies or retinal degenerative disease. The rAAV, preferablysuspended in a physiologically compatible carrier, diluent, excipientand/or adjuvant, may be administered to a desired subject includingwithout limitation, a cat, dog, or other non-human mammalian subject.This method comprises administering to a subject in need thereof any ofthe nucleic acid sequences, expression cassettes, rAAV genomes,plasmids, vectors or rAAV vectors or compositions containing them. Inone embodiment, the composition is delivered subretinally. In anotherembodiment, the composition is delivered intravitreally. In stillanother embodiment, the composition is delivered using a combination ofadministrative routes suitable for treatment of ocular diseases, and mayalso involve administration via the palpebral vein or other intravenousor conventional administration routes.

For use in these methods, the volume and viral titer of each dosage isdetermined individually, as further described herein, and may be thesame or different from other treatments performed in the same, orcontralateral, eye. In another embodiment, a single, larger volumetreatment is made in order to treat the entire eye. The dosages,administrations and regimens may be determined by the attendingphysician given the teachings of this specification.

In one embodiment, the composition is administered in a single dosageselected from those above listed in a single affected eye. In anotherembodiment, the composition is administered as a single dosage selectedfrom those above listed in a both affected eyes, either simultaneouslyor sequentially. Sequential administration may imply a time gap ofadministration from one eye to another from intervals of minutes, hours,days, weeks or months. In another embodiment, the method involvesadministering the compositions to an eye two or more dosages (e.g.,split dosages).

In still other embodiments, the compositions described herein may bedelivered in a single composition or multiple compositions. Optionally,two or more different AAV may be delivered, or multiple viruses [see,e.g., WO 2011/126808 and WO 2013/049493]. In another embodiment,multiple viruses may contain different replication-defective viruses(e.g., AAV and adenovirus).

In certain embodiments of the invention it is desirable to performnon-invasive retinal imaging and functional studies to identify areas ofthe rod and cone photoreceptors to be targeted for therapy. In theseembodiments, clinical diagnostic tests are employed to determine theprecise location(s) for one or more subretinal injection(s). These testsmay include electroretinography (ERG), perimetry, topographical mappingof the layers of the retina and measurement of the thickness of itslayers by means of confocal scanning laser ophthalmoscopy (cSLO) andoptical coherence tomography (OCT), topographical mapping of conedensity via adaptive optics (AO), functional eye exam, etc, dependingupon the species of the subject being treated, their physical status andhealth and the dosage. In view of the imaging and functional studies, insome embodiments of the invention one or more injections are performedin the same eye in order to target different areas of the affected eye.The volume and viral titer of each injection is determined individually,as further described below, and may be the same or different from otherinjections performed in the same, or contralateral, eye. In anotherembodiment, a single, larger volume injection is made in order to treatthe entire eye. In one embodiment, the volume and concentration of therAAV composition is selected so that only the region of damaged rod andcone receptors is impacted. In another embodiment, the volume and/orconcentration of the rAAV composition is a greater amount, in orderreach larger portions of the eye, including non-damaged photoreceptors.

In one embodiment of the methods described herein, a one-timeintra-ocular delivery of a composition such as those described herein,e.g., an AAV delivery of an optimized RdCVF or RdCVFL cassette, isuseful in preventing vision loss and blindness in millions ofindividuals affected with such ocular disorders or multi-systemicdiseases without regard to genotype or environmental exposure.

Thus, in one embodiment, the composition is administered before diseaseonset. In another embodiment, the composition is administered prior tothe initiation of vision impairment or loss. In another embodiment, thecomposition is administered after initiation of vision impairment orloss. In yet another embodiment, the composition is administered whenless than 90% of the rod and/or cones or photoreceptors are functioningor remaining, as compared to a non-diseased eye.

In another embodiment, the method includes performing additionalstudies, e.g., functional and imaging studies to determine the efficacyof the treatment. For examination in animals, such tests include retinaland visual function assessment via electroretinograms (ERGs) looking atrod and cone photoreceptor function, optokinetic nystagmus,pupillometry, water maze testing, light-dark preference histology(retinal thickness, rows of nuclei in the outer nuclear layer,immunofluorescence to document transgene expression, cone photoreceptorcounting, staining of retinal sections with peanut agglutinin—whichidentifies cone photoreceptor sheaths). Other suitable tests of efficacyare sampling of anterior chamber fluid to document presence of the RdCVFand RdCVFL transgenic proteins.

Specifically for human subjects, following administration of a dosage ofa compositions described in this specification, the subject is testedfor efficacy of treatment using electroretinograms (ERGs) to examine rodand cone photoreceptor function, pupillometry visual acuity, contrastsensitivity color vision testing, visual field testing (Humphrey visualfields/Goldmann visual fields), perimetry mobility test (obstaclecourse), and reading speed test. Other useful post-treatment efficacytest to which the subject is exposed following treatment with apharmaceutical composition described herein are functional magneticresonance imaging (fMRD, full-field light sensitivity testing, retinalstructure studies including optical coherence tomography, fundusphotography, fundus autofluorescence, adaptive optics scanning, and/orlaser ophthalmoscopy. These and other efficacy tests are described inU.S. Pat. No. 8,147,823; in co-pending International patent applicationpublication WO 2014/011210 or WO 2014/124282, incorporated by reference.

In yet another embodiment, any of the above described methods isperformed in combination with another, or secondary, therapy. In stillother embodiments, the methods of treatment of these ocular diseasesinvolve treating the subject with the composition described in detailherein in combination with another therapy, such as antibiotictreatment, palliative treatment for pain, and the like. The additionaltherapy may be any now known, or as yet unknown, therapy which helpsprevent, arrest or ameliorate these mutations or defects or any of theeffects associated therewith. The secondary therapy can be administeredbefore, concurrent with, or after administration of the compositionsdescribed above. In one embodiment, a secondary therapy involvesnon-specific approaches for maintaining the health of the retinal cells,such as administration of neurotrophic factors, anti-oxidants,anti-apoptotic agents. The non-specific approaches are achieved throughinjection of proteins, recombinant DNA, recombinant viral vectors, stemcells, fetal tissue, or genetically modified cells. The latter couldinclude genetically modified cells that are encapsulated.

In one embodiment, a method of generating a recombinant rAAV comprisesobtaining a plasmid containing a rAAV genome as described above andculturing a packaging cell carrying the plasmid in the presence ofsufficient viral sequences to permit packaging of the AAV viral genomeinto an infectious AAV envelope or capsid. Specific methods of rAAVvector generation are described above and may be employed in generatinga rAAV vector that can deliver one or more of the codon optimized RdCVFLor RdCVF in the expression cassettes and genomes described above and inthe examples below.

The following examples disclose specific embodiments of the nucleic acidsequences, expression cassettes, rAAV genome and viral vectors for usein treating the ocular diseases specified herein. These specificembodiments illustrate various aspects of the invention. These examplesshould be construed to encompass any and all variations that becomeevident as a result of the teaching provided herein.

EXAMPLE 1 Codon Optimized Sequences

The nucleic acid sequence SEQ ID NO: 1 (FIG. 1) encoding codon optimizedhuman RdCVFL was generated to add N-terminal restriction site SfiI and Cterminal restriction site Bg1II, as well as Kozak sequences. The openreading frame (ORF) of codon optimized SEQ ID NO:1 differs from thenative sequence by 17%, i.e., it shares only 83% identity with nativehRdCVF, as shown in FIG. 3.

The nucleic acid sequence SEQ ID NO: 2 (FIG. 1) encoding codon optimizedhuman RdCVF was generated to add N-terminal restriction site Notl and Cterminal restriction site BM, as well as Kozak sequences. The ORF ofcodon optimized SEQ ID NO:2 differs from the native sequence by 17%,sharing only 83% identity with native hRdCVF, as shown in FIG. 4.

EXAMPLE 2 Construction of P853

SEQ ID NO: 2 was cloned into an expression vector under the control of achicken-beta actin promoter with CMV enhancer, the promoter truncated by390 nucleotides. SEQ ID NO: 1 was cloned into the same cassette underthe control of a second copy of the same promoter. The expressionconstruct was flanked by AAV2 ITRs thus forming the p853 rAAV genomep853 (See FIG. 5). This rAAV genome was then inserted into a proviralplasmid, p618 containing a lambda stuffer sequence (see InternationalPatent Application Publication No. WO2012/158757A1), thereby generatingthe proviral plasmid p853 which permits expression of both the long andshort RdCVF proteins in a single vector.

The features of the rAAV genome of FIG. 5 SEQ ID NO: 4 and the p853plasmid pAAV.CMV.CBA.hRdCVF.CMV.CBA.hRDCVFL.SYNITR.Long SEQ ID NO: 7containing the rAAV genome of FIG. 5 and other plasmid sequences aredescribed below in Table 1, with reference to the nucleotide positions(Nts) in each sequence.

TABLE 1 SEQUENCES rAAV Genome Cassette Nts of SEQ IDpAAV.CMV.CBA.hRdCVF.CMV.CBA.hRdCVFL.SYNITR.long Sequence Feature NO: 4Nts of SEQ ID NO: 7 Kan^(R) Complement —  9-803 B1 B2 T1 Txn —  988-1162Terminator Complement pTR — 1063-1079 5′ ITR (D Segment)  17-1461253-1382 (129-146) (1365-1382) Promoter Delta 390  207-1478 1443-2714hRdCVF 1479-1826 2715-3062 Poly A 1821-2047 3057-3283 Terminator2042-2479 3278 -3715  (Promoter) With (2490-3761) (3726-4997) FlankingRestriction 2474-3774 3710-5010 Sites hRDCVFL 3762-4426 4998-5662 (Bgl)Poly A 4427-4643 5663-5879 3′ ITR (D Segment) 4691-4820 5927-6056(4691-4708) (5927-5944) pTF3 Complement — 6241-6266 BLA Txn Terminator —6150-6450 RPN Txn Terminator — 6457-6570 Lambda Stuffer —  6586-11652pUC Ori Complement — 11813-12616

EXAMPLE 3 Construction of rAAV Co-Expressing hRdCVF and hRdCVFL

The rAAV genome from this p853 proviral plasmid SEQ ID NO: 7 is packagedin a selected AAV capsid by culturing a packaging cell carrying theplasmid in the presence of sufficient viral sequences to permitpackaging of the AAV genome into an infectious AAV envelope or capsid.In one embodiment, a method for producing the rAAV involves packaging ina stable rep and cap expressing mammalian host packaging cell line (suchas B-50 as described in International Patent Application Publication No.WO 99/15685) with the adenovirus E1, E2a, and E4ORF6 DNA. Iodixanolgradient purification is followed by herparin-sepharose agarose columnchromatography. Vector titers are determined using an infectious centerassay.

Recombinant AAV.CMV.CBA.hRdCVF.CMV.CBA.hRdCVFL. SYNITR.Long viruspreparations are prepared in and combined to a desired total volume.

Still other methods of producing such rAAV particles involve use of aninsect cell packaging cell line, such as described in Smith et al, ref11, cited below.

The AAV.CMV.CBA.hRdCVF.CMV.CBA.hRdCVFL.SYNITR.Long viral particles aresuspended in a suitable excipient, such as 180 mM NaCl, 10 mM NaPi,pH7.3, containing 0.0001%-0.01% Pluronic F68 (PF68). The composition ofthe saline component ranges from 160 mM to 180 mM NaCl. Other buffersare useful in such compositions, including HEPEs, sodium bicarbonate,TRIS, or 0.9% NaCl solution.

Several preparations of the rAAV are combined to a desired total volume.In one embodiment, a total volume is a dosage of 1×0¹¹ GC in a volume of300 microliters of buffer. Contaminating helper adenovirus and nativeAAV, assayed by serial dilution cytopathic effect or infectious centerassay, respectively are anticipated to be less than one or multiplesorders of magnitude lower than vector AAV.

EXAMPLE 4 Construction of rAAV Expressing 2XRdCVF

A native short form of RdCVF (or the codon optimized SEQ ID NO: 2) wascloned into an expression vector under the control of a chicken-betaactin promoter with CMV enhancer, the promoter truncated by 390nucleotides. A second copy of the native sequence of RdCVF (or the codonoptimized SEQ ID NO: 2) was cloned into the same cassette under thecontrol of a second copy of the same promoter. The expression constructwas flanked by AAV2 ITRs thus forming the rAAV genome 2×RdCVF. The rAAVgenome with native RdCVF sequences is reported in SEQ ID NO: 5. The rAAVgenome with codon optimized sequences is reported as SEQ ID NO: 8.Either of these rAAV genomes was then inserted into a proviral plasmid,p617 (see International Patent Application Publication No.WO2012/158757A1), thereby generating the proviral plasmid SEQ ID NO: 6(with native RdCVF) or the proviral plasmid p857 of SEQ ID NO: 9 (codonoptimized RdCVF) which permits expression of two copies of the shortRdCVF protein in a single vector.

The features of the rAAV genome 2×RdCVF containing the native sequencesSEQ ID NO: 5) and the plasmid calledpAAV.CMV.CBA.delta390.hRdCVFL.2×.synITR.long—native RdCVF (SEQ ID NO: 6)are described below in Table 2A, with reference to the nucleotidepositions (Nts) in each sequence.

TABLE 2A SEQUENCES rAAV genome pAAV.CMV.CBA.delta- 2xRdCVF -390.hRdCVF1.2x.- native synITR.long - sequence native sequences Nts OfNts Of SEQ ID SEQ ID Sequence Feature NO: 5 NO: 6 Kan^(R) complement 9-100  9-803 B1 B2 T1 Txn Terminator —  988-1162 Complement pTR —1063-1079 5 ITR (D segment) 117-246 1253-1382 (229-246) (1365-1382)Promoter Delta 390  307-1578 1443-2714 hRdCVF 1591-1935 2727-3071 Poly A1940-2161 3076-3297 pTF3 complement 2337-2362 3473-3498 Bla TxnTerminator 2246-2546 3382-3682 Rpn Txn Terminator 2553-2666 3689-3802Promoter 2697-3968 3833-5104 hRDCVF 3981-4325 5117-5461 Poly A 4330-45515466-5687 3′ ITR 4599-4728 5735-5864 BLA Txn Terminator — 5958-6258 pTF3Complement — 6049-6074 Rpn Txn Terminator — 6265-6378 Lambda Stuffer — 6394-11460 pUC Ori Complement — 11621-12424

The features of the rAAV genome 2×RdCVF containing the codon optimizedsequences of RdCVF (SEQ ID NO: 8) and p857opt plasmid calledpAAV.CMV.CBA.delta390.hRdCVFL.2×.synITR.long (SEQ ID NO: 9) aredescribed below in Table 2B, with reference to the nucleotide positions(Nts) in each sequence.

TABLE 2B p857opt SEQUENCES pAAV.CMV.CBA.delta- rAAV genome390.hRdCVF1.2x.- cassette synITR.long Nts Of Nts Of SEQ ID SEQ IDSequence Feature NO: 8 NO: 9 Kan^(R) complement  9-100  9-803 B1 B2 T1Txn Terminator —  988-1162 Complement pTR — 1063-1079 5′ ITR (D segment)117-246 1253-1382 (229-246) (1365-1382) Promoter Delta 390  307-15781443-2714 hoptRdCVF 1579-1926 2715-3062 Poly A 1931-2152 3067-3288 pTF3complement 2328-2353 3464-3489 Bla Txn Terminator 2237-2537 3373-3673Rpn Txn Terminator 2544-2657 3680-3793 Promoter 2688-3959 3824-5095hoptRDCVF 3960-4307 5096-5443 Poly A 4312-4533 5448-5669 3′ ITR4581-4710 5717-5846 BLA Txn Terminator — 5940-6240 pTF3 Complement —6031-6056 Rpn Txn Terminator — 6247-6360 Lambda Stuffer —  6376-11442pUC Ori Complement — 11603-12406

EXAMPLE 5 Construction of rAAV Co-Expressing Two Copies of hRDCVF

The rAAV genome from the proviral plasmids of SEQ ID NO: 8 or SEQ ID NO:9 is packaged in a selected AAV capsid by culturing a packaging cellcarrying the plasmid in the presence of sufficient viral sequences topermit packaging of the AAV genome into an infectious AAV envelope orcapsid. In one embodiment, a method for producing the rAAV involvespackaging in a stable rep and cap expressing mammalian host packagingcell line (such as B-50 as described in International Patent ApplicationPublication No. WO 99/15685) with the adenovirus E1, E2a, and E4ORF6DNA. Iodixanol gradient purification is followed by heparin-sepharoseagarose column chromatography. Vector titers are determined using aninfectious center assay.

AAV.CMV.CBA.delta390.hRdCVF1.2×.synITR.long virus (native or codonoptimized) preparations are prepared and suspended in a suitableexcipient, such as 180 mM NaCl, 10 mM NaPi, pH7.3, containing0.0001%-0.01% Pluronic F68 (PF68). The composition of the salinecomponent ranges from 160 mM to 180 mM NaCl. Other buffers are useful insuch compositions, including HEPEs, sodium bicarbonate, TRIS, or 0.9%NaCl solution.

Several preparations of the rAAV are combined to a desired total volume.In one embodiment, a total volume is 1.0 to 10⁵ ml containing either adose of 2.3×10¹¹ infectious particles or viral genomes or aconcentration of 2.3×10¹¹ infectious particles or viral genomes/ml.Contaminating helper adenovirus and native AAV, assayed by serialdilution cytopathic effect or infectious center assay, respectively areanticipated to be orders of magnitude lower than vector AAV.

In a similar manner an rAAV containing tandem expression of two copiesof the long form of RdCVF (2×RdCVFL) and corresponding plasmid aregenerated.

Still other methods of producing such rAAV particles involve use of aninsect cell packaging cell line, such as described in Smith et al, ref11, cited below.

EXAMPLE 6 In Vitro And In Vivo Tests

The suspension of rAAV particles described in Examples 1 through 5 areemployed to transduce target cell cultures in vitro, such as in mice orchicken retinal cell cultures, at multiplicities of infection (MOI)ranging from 10³ to 10⁶ rAAV viral particles per cell. Survival countsof cone photoreceptors from such species are counted to demonstrate theefficacy of gene transfer. Other suitable techniques for determiningefficacy in such in vitro models are RT-PCR, immunocytochemistry,immunohisto-chemistry, and Western blot analysis.

The rAAV particles are also employed to transduce cells of the murine orother mammalian (e.g., canine or feline) retina after administration bysubretinal injection of 10¹¹-10¹³ viral particles or 10¹¹-10¹³ viralparticles /ml buffer. Expression of both hRdCVFL and hRdCVF together intransduced cells or retinas or expression of two copies of hRdCVF isassessed by retinal and visual function. These functions may be examinedin animals using one or more of the techniques: electroretinograms(ERGs) looking at rod and (especially) cone photoreceptor function,optokinetic nystagmus, pupillometry, water maze testing, light-darkpreference histology (retinal thickness, rows of nuclei in the outernuclear layer, immunofluorescence to document transgene expression, conephotoreceptor counting, staining of retinal sections with peanutagglutinin—which identifies cone photoreceptor sheaths). Additionally,sampling of anterior chamber fluid is used to document the presence ofthe RdCVF transgenic protein.

EXAMPLE 7 Efficacy In Human Subjects

The rAAV particles are also employed to transduce cells of humansubject's retina after administration by subretinal injection of10¹⁰-10¹² GC or viral particles in a suspension in a suitable bufferedcarrier. Expression of both hRdCVFL and hRdCVF together in transducedcells or retinas or expression of two copies of hRdCVF is assessed byretinal and visual function.

These functions may be examined in humans using one or more of thetechniques: electroretinograms (ERGs) looking at rod and conephotoreceptor function pupillometry visual acuity contrast sensitivitycolor vision testing visual field testing (Humphrey visualfields/Goldmann visual fields) perimetry mobility test (obstacle course)reading speed test. Other useful tests include functional magneticresonance imaging (fMRI) full-field light sensitivity testing, retinalstructure studies including optical coherence tomography, fundusphotography, fundus autofluorescence, adaptive optics and scanning laserophthalmoscopy.

Methods of use of these recombinant viruses are introduced into humansubjects and evaluated by techniques, such as described in the examplesdescribed in published international applications WO2016/185037 andWO2016/185242, incorporated by reference herein.

Each and every patent, patent application, and publication, includingwebsites cited throughout specification, as well as U.S. provisionalpatent application No. 62/275006, are incorporated herein by reference.Similarly, the SEQ ID NOs which are referenced herein and which appearin the appended Sequence Listing are incorporated by reference. Whilethe invention has been described with reference to particularembodiments, it will be appreciated that modifications can be madewithout departing from the spirit of the invention. Such modificationsare intended to fall within the scope of the appended claims.

TABLE 3 (Sequence Listing Free Text) The following information isprovided for sequences containing free text under numeric identifier<223>. SEQ ID NO: (containing free text) Free text under <223> 4 rAAVgenome with ITRs flanking an expression cassette containing RdCVFsequences and promoters 4 AAV 5′ ITR 4 hRdCVF short form codon optimized4 hRdCVFL long form codon optimized 4 AAV 3′ ITR 5 p857 rAAV genome withtwo copies of RdCVF and promoters 5 KanR complement 5 AAV 5′ ITR 5 humanRdCVF native short form 5 pTF3 complement 5 human RdCVF native shortform second copy 5 AAV 3′ ITR 6 Plasmid containing the p857 rAAV genomecontaining native 2xRdCVF 6 KanR complement 6 pTR 6 AAV 5 ITR 6 B1 B2 T1Txn Terminator Complement 6 hRdCVF (native short form) 6 pTF3 complement6 hRdCVF native short form second copy 6 AAV 3′ ITR 6 pTF3 Complement 6Lambda Stuffer 6 pUC Ori Complement 7 Plasmid containing the rAAV genomeof p853 7 KanR complement 7 B1 B2 T1 Txn Terminator Complement 7 pTR 7AAV 5′ ITR 7 human hRdCVF short form optimized sequence 7 hRDCVFLoptimized sequence long form 7 AAV 3′ ITR 7 pTF3 Complement 7 LambdaStuffer 7 pUC Ori Complement 8 rAAV genome expressing two copies ofhuman RdCVF codon optimized short form sequences 8 KanR complement 8 AAV5′ ITR 8 human optimized RdCVF short form 8 pTF3 complement 8 humanoptimized RdCVF short form second copy 8 AAV 3′ ITR 9 plasmid expressingrAAV genome with two copies of optimized human RdCVF short form 9 KanRcomplement 9 pTF3 complement 9 B1 B2 T1 Txn Terminator Complement 9 pTR9 5′ AAV ITR 9 human optimized RdCVF short form 9 human optimized RdCVFshort form second copy 9 AAV 3′ ITR 9 pTF3 Complement 9 Lambda Stuffer 9pUC Ori Complement

REFERENCES

-   1. U.S. Pat. No. 7,795,387-   2. U.S. Pat. No. 8, 114,849-   3. U.S. Pat. No. 8,394,756-   4. U.S. Pat. No. 8,518,695-   5. U.S. Pat. No. 8,957,043-   6. US Patent Appin Publication No. 2009062188A1-   7. Natalia Caporale et al, July 2011, LiGluR Restores Visual    Responses in Rodent Models of Inherited Blindness, Mol. Therapy,    19(7):1212-1219-   8. Kotin, R M, April 2011 Large-scale recombinant adeno-associated    virus production., Hu. Mol. Genet, 20(1): R1-R6-   9. Byrne et al, January 2015, “Viral-mediated RdCVF and RdCVFL    expression protects cone and rod photoreceptors in retinal    degeneration”, J. Clin. Invest.,125(1):105-116-   10. Maguire, A M et al., October 2009 Age-dependent effects of RPE65    gene therapy for Leber's congenital amaurosis: a phase 1    dose-escalation trial. Lancet, DOI:10.1016/S0140-6736(09)61836-5-   11. Smith, R H et al, June 2009 A Simplified Baculovirus-AAV    Expression Vector System Coupled With One-step Affinity Purification    Yields High-titer rAAV Stocks From Insect Cells, Mol. Ther., 17(11):    1889-1896-   12. Bennicelli J et al, March 2008 Reversal of Blindness in Animal    Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated    Gene Transfer, Mol. Ther., 16(3):458-465-   13. International Patent Application Publication No. WO2013/063383-   14. International Patent Application Publication No. WO2016/185037-   15. International Patent Application Publication No. WO2016/185242

1. An expression cassette comprising one or more of: (a) a codonoptimized nucleic acid sequence SEQ ID NO: 1 that encodes RdCVFL, or (b)a codon optimized nucleic acid sequence SEQ ID NO: 2 that encodes RdCVF;(c) a codon optimized nucleic acid sequence SEQ ID NO: 1 that encodesRdCVFL SEQ ID NO: 1 and a codon optimized nucleic acid sequence SEQ IDNO: 2 that encodes RdCVF, or (d) two copies of a codon optimized nucleicacid sequence SEQ ID NO: 2 that encodes two copies of RdCVF.
 2. Theexpression cassette according to claim 1, wherein each said codonoptimized sequence is operatively associated with an expression controlsequence that directs expression of each codon optimized nucleic acidsequence that encodes RdCVF or RdCVFL in a host cell.
 3. A codonoptimized, engineered nucleic acid sequence encoding human RdCVFL orRdCVF comprising SEQ ID NO: 1 or SEQ ID NO:
 2. 4. A recombinant AAVgenome, wherein said rAAV genome comprises AAV inverted terminal repeatsequences flanking an expression cassette of claim
 1. 5. A vector orplasmid comprising the expression cassette of claim
 1. 6. A recombinantadeno-associated virus (AAV) comprising an AAV capsid and an recombinantAAV genome, wherein said rAAV genome comprises AAV inverted terminalrepeat sequences and an expression cassette comprising at least one ofSEQ ID NO: 1 or SEQ ID NO: 2 and expression control sequences thatdirect expression of the encoded proteins in a host cell.
 7. The rAAVaccording to claim 6, comprising AAV inverted terminal repeat sequencesflanking two expression cassettes or transgenes in tandem, eachexpression cassette or transgene comprising a nucleic acid sequence SEQID NO: 2 that encodes RdCVF or a nucleic acid sequence SEQ ID NO: 1 thatencodes RdCVFL.
 8. The rAAV according to claim 7, wherein eachexpression cassette or transgene comprises a nucleic acid sequence SEQID NO: 2 that encodes the short isoform of RdCVF.
 9. The rAAV accordingto claim 7, wherein each transgene comprises a nucleic acid sequence SEQID NO: 1 that encodes the long isoform of RdCVFL.
 10. The rAAV accordingto claim 7, wherein one expression cassette or transgene comprises anucleic acid sequence SEQ ID NO: 2 that encodes RdCVF and the otherexpression cassette or transgene comprises a nucleic acid sequence SEQID NO: 1 that encodes RdCVFL.
 11. The rAAV of claim 6, comprising a 5′AAV ITR sequence, a CMV/CBA promoter, a first nucleic acid sequence SEQID NO. 1 or 2 that encodes RdCVFL or RdCVF, a polyadenylation sequence,transcriptional terminator sequences, another CMV/CBA promoter, a secondnucleic acid sequence SEQ ID NO: 1 or 2 that encodes RdCVFL or RdCVF, asecond polyadenylation sequence and a 3′ AAV ITR sequence.
 12. A hostcell comprising the expression cassette of claim
 1. 13. A compositioncomprising an engineered nucleic acid molecule useful in the treatmentof an ocular disease comprising a nucleic acid sequence SEQ ID NO: 1encoding RdCVFL, the sequence SEQ ID NO: 2 encoding RdCVF, both SEQ IDNO: 1 and SEQ ID NO: 2, or two copies of SEQ ID NO: 1 or two copies ofSEQ ID NO: 2, each protein encoding nucleic acid sequence under thecontrol of expression control sequences which direct expression ofRdCVFL or RdCVF in ocular cells; and a carrier suitable for delivery tothe eye of a subject.
 14. A composition comprising a pharmaceuticallyacceptable carrier suitable for delivery to the eye and the recombinantvirus of claim
 6. 15. A method for treating, retarding or haltingprogression of an ocular disease or blinding disease in a mammaliansubject, said method comprising administering the composition of claim14 to a subject in need thereof.
 16. The method according to claim 15,wherein said composition is administered subretinally or intravitreally.17. The method according to claim 15, wherein said disease is a rod-conedystrophy or retinal degeneration.
 18. (canceled).